Methods and use of compounds that bind to HER2/neu receptor complex

ABSTRACT

This application describes pharmaceutical compositions, commercial packages, and methods for inhibiting cell proliferative disorders, especially those disorders, including Her2 related cancers, characterized by overactivity and/or inappropriate activity of a receptor tyrosine kinase, and methods for imaging an HER-2 expressing tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2011/038044, filed May 26, 2011, which claims the benefit of U.S.Provisional Application No. 61/350,700 filed Jun. 2, 2010, thedisclosures of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

This invention is in the fields of cancer diagnosis and therapy. Moreparticularly it concerns compounds which are useful agents forinhibiting cell proliferative disorders, especially those disorders,including Her2 related cancers, characterized by over activity and/orinappropriate activity of a receptor tyrosine kinase and methods fortreating these disorders. It also describes methods for measuring andmonitoring growth of tumors.

BACKGROUND

Cancer affects approximately 10 million people globally and it isexpected to increase to 15 million by 2020. Worldwide approximately 23million people are living with cancer and about 5.2 million people die.Breast and lung cancer account for 25% of cancer incidences.

Expression of Human Epidermal growth factor receptor 2 (or “Her2”) inbreast cancer, prostate cancer, lung cancer, gastric cancer and the likehas been reported and Her2 is considered to be involved in the growth ofthese cancers. For example, Slamon et al., Science 235, 177 (1987)demonstrated that about 30% of primary human breast carcinoma tumorscontained an amplified Her2 gene. More recently, it has been reportedthat about 25% of primary prostate cancer is Her2 expression positive,and the percentage increases along with the progression of the cancer(Journal of the National Cancer Institute, Vol. 92, No. 23, pp.1918-1925 (2000)). Similarly, 4-27% of lung cancer cases are reported tooverexpress Her2/neu (ErbB2). Overexpression of HER2/neu and other erbBreceptors is associated with increased disease recurrence and leads to apoor prognosis in such cancers.

Many other diseased states are also characterized by the uncontrolledreproduction of cells. These diseased states involve a variety of celltypes and, in addition to leukemia and cancer, include psoriasis,atherosclerosis and restenosis injuries. The inhibition of tyrosinekinase is believed to have utility in the control of uncontrolledcellular reproduction, i.e., cellular proliferative disorders

Her2 is also known as neu, ErbB-2, or ERBB2. Her2/neu (ErbB2), a memberof the type I transmembrane receptor tyrosine kinase family, regulatescell differentiation and growth during embryogenesis and breastdevelopment. This oncogene was first obtained from rat neuroglioblastomainduced by a chemical carcinogenic substance was found to encode aprotein belonging to the EGF receptor family, and the relationship withwhich was suggested. Thereafter, an neu human homologue was isolated andnamed as ERBB2 or Her2 based on the similarity to an EGF receptor, ERBB(avian erythroblastosis oncogene B). The oncogene later found to codefor EGFR (Epidermal Growth Factor Receptor). Gene cloning subsequentlydetermined that neu, Her2, and erbB2 are species variants of oneanother. (Slamon, D. J., et al., Science, 235:177 182, 1987).

ErbB receptors or epidermal growth factor receptors (EGFR) play animportant role in a variety of signal transduction pathways that promotecell differentiation, growth, proliferation, and migration. Interactionbetween Erb receptors allows ErbB2/Her2 to participate in effectivesignaling. In particular, EGFR and Her2 have been implicated in thedevelopment of human cancer, hence Her2 represents an attractivetherapeutic target against Her2 positive cancers.

Her2 is a cell membrane surface bound receptor tyrosine kinase, normallyinvolved in the signal transduction pathways leading to cellproliferation, differentiation, and migration. Co-expression of Her2/neuand other members of erbB in normal cells promotes formation of dimercomplexes, which cause malignant cell transformation. Olayioye M A(2001). “Update on Her-2 as a target for cancer therapy: intracellularsignaling pathways of ErbB2/Her-2 and family members,” Breast CancerRes. 3(6): 385-389.

Her2 hardly expresses in normal tissues. Therefore, an Her2 selectivetherapeutic drug would be cancer selective, with the reduced toxicity orextremely few side effects. This means that an extreme safe and highlyversatile treatment method could be provided, which is strikinglydifferent from conventional cancer chemotherapeutic agents.

Currently, the only drugs that can disable both Her2 and EGFr function(Trastuzumab or Herceptin, Pertuzumab, and similar humanized monoclonalantibodies) are targeted to receptor tyrosine kinase in the cytoplasmicdomains. Small molecule tyrosine kinase inhibitors such as thequinazolinamine, Lapatinib(N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]-2-furyl]quinazolin-4-amine),which compete for ATP binding sites on the tyrosine kinase domain ofreceptors are also being used to treat Her2 positive tumors.

Attempts have also been made to identify other small molecules which actas tyrosine kinase inhibitors, and other compounds inhibiting areceptor-type tyrosine kinase (including Her2/EGFR kinase) include fusedheterocyclic compounds such as compounds generally depicted as

(see, for example, WO97/13771, WO98/02437, WO00/44728, U.S. Pat. No.6,596,878, US 2005/0148607, and US 2008/0214584), quinazolinederivatives such as compounds generally depicted as

(see, for example, WO02/02552, WO01/98277, WO03/049740 and WO03/050108,U.S. Pat. No. 6,596,878), thienopyrimidine derivatives such as compoundsgenerally depicted as

(see, for example, WO03/053446, U.S. Pat. No. 7,300,935), thienylderivatives such as compounds generally depicted as

(see for example, U.S. Pat. No. 5,710,173), aromatic azole derivativessuch as compounds generally depicted as

(see, for example, WO01/77107, WO03/031442, U.S. Pat. Nos. 6,716,863 and6,984,653), and the like are known.

Further, bis monocyclic, bicyclic or heterocyclic aryl compounds such ascompounds generally depicted as

(see, for example, WO 92/20642), vinylene-azaindole derivatives such ascompounds generally depicted as

(see, for example, WO94/14808), azaindoles such as compounds generallydepicted as

(see, for example, WO03/000688 and WO96/000226) and1-cycloproppyl-4-pyridyl-quinolones such as compounds generally depictedas

(see, for example, U.S. Pat. No. 5,330,992) have been describedgenerally as tyrosine kinase inhibitors. Styryl compounds such ascompounds generally depicted as

(see, for example, U.S. Pat. Nos. 5,217,999, 5,596,878),styryl-substituted pyridyl compounds such as compounds generallydepicted as

(see, for example, U.S. Pat. No. 5,302,606), tyrphostin-like compoundssuch as compounds generally depicted as

(see, for example, U.S. Pat. No. 6,225,346), seleoindoles and selenidessuch as compounds generally depicted as

(see, for example, WO94/03427), 1H-pyrrolo[2,3-b]pyridines such ascompounds generally depicted as

(see, for example, WO01/098299), tricyclic polyhydroxylic compounds suchas compounds generally depicted as

(see, for example, WO92/21660), 2-pyrazolin-5-ones such as compoundsgenerally depicted as

(see, for example, WO 01/01921), and benzylphosphonic acid compoundssuch as compounds generally depicted as

(see, for example, WO91/15495) have been described as compounds for useas tyrosine kinase inhibitors for use in the treatment of cancer.

While drugs targeted to the receptor-type tyrosine kinase enzyme haveshown promise in treating some cancers, the concern remains that chronicuse of such agents that depend on tyrosine kinase domain receptors maylead to acquired resistance in patients. Indeed, such resistance may beseen within 12 months of the treatment with such agents. It is believedthat these reflect the fact that kinase domains can alter theirstructure and become resistant over regular use. Beyond this, thestructure of kinase domain is ubiquitous and highly conserved and longuse of kinase inhibitors can inhibit other kinases involved in thedevelopment of cells/organs. Thus, these kinase domain targeted drugsmay be unsuitable for small children with cancers.

SUMMARY

The present invention relates to use of small molecule compounds tomodulate or inhibit the activity of a receptor tyrosine kinase.

In some embodiments, the invention teaches a method of inhibiting a cellproliferative disorder characterized by over-activity and/orinappropriate activity of a receptor, especially an Her2 receptor,comprising administering to a patient in need of such treatment apharmaceutically effective amount of a compound of Formula (A), or apharmaceutically acceptable salt or prodrug thereof:

where n is 1-5 and B is independently alkyl, hydroxy, halo,perfluoromethyl, carboxylic acid, ester, amide, substituted alkoxy,substituted thiol, substituted acyl, nitrile, or substituted aminogroup.

In other embodiments, the invention teaches a method of inhibiting acell proliferative disorder characterized by over-activity and/orinappropriate activity of a protein tyrosine kinase portion of areceptor, especially an Her2 receptor, comprising administering to apatient in need of such treatment a pharmaceutically effective amount ofa compound of Formula (I), or a pharmaceutically acceptable salt orprodrug thereof:

or its tautomer

wherein X is independently at each occurrence a hydrogen, halo, hydroxy,carboxylic acid, ester, amide, substituted alkoxy, substituted thiol,substituted acyl, nitrile, or substituted amino group.

In still other embodiments, the compound is of Formula (I) wherein X ishalo, preferably chloro or iodo, most preferably chloro.

In still other embodiments, the compound of Formula (I) is

In further embodiments, the invention discloses a method of treating apatient having a disease characterized by over-activity and/orinappropriate activity of a protein tyrosine kinase portion of areceptor, especially an Her2 receptor, comprising the step ofadministering to said patient a pharmaceutically effective amount of acompound having the Formula (I), of any of the previously cited chemicalembodiments.

In still further embodiments, the invention teaches the use of acompound of any of the preceding chemical embodiments of Formula (I) forthe preparation of a medicament for the inhibition of a cellproliferative disorders characterized by over-activity and/orinappropriate activity of a protein tyrosine kinase portion of areceptor, especially an Her2 receptor, and in still other embodiments,the invention discloses a pharmaceutical composition comprising acompound of Formula (I) in an amount effective to inhibit a cellproliferative disorder characterized by over-activity and/orinappropriate activity of a protein tyrosine kinase portion of areceptor, especially an Her2 receptor. Each pharmaceutical compoundembodiment may be part of a commercial package, further comprising atleast one anti-cancer agent and written instructions as to the use ofthe medicament.

In each of these embodiments, the receptor can include an erbB receptor,preferably an epidermal growth factor receptor (EGFR), or aplatelet-derived growth factor receptor, and/or the receptor is Her2,Her3, or Her4, such that the aim of the method or composition is toinhibit the overexpression or activation of Her2, Her3, or Her4 and/orEGFR, thereby inhibiting associated diseases such as cancer,angiogenesis associated with the growth of cancer or sarcoma,angiogenesis associated with cancer metastasis, angiogenesis associatedwith diabetic retinopathy, arteriosclerosis, restenosis, or psoriasis.Exemplary cancers include breast, prostate, lung, pancreatic, ovarian,or stomach cancers.

In other embodiments, the patient being treated or to whom thecomposition is to be applied is a human.

This invention can be applied to treat cell proliferative disorders,such as cancers characterized by the over-activity or inappropriateactivity of Her2/neu, when used alone or in combination withchemotherapy (e.g., cytotoxic chemotherapy or radiation or both) to haltresistance to currently available drugs. The invention may also beapplied in combination with administering a pharmaceutically effectiveamount of an anti-cancer agent or performing a non-drug therapy or bothon the patient.

The invention provides a novel class of Her2 and EGFR inhibitors thatmay halt the resistance to current tyrosine kinase inhibitors henceincrease treatment efficacy, and as such provide an alternative to theuse of current tyrosine kinase inhibitors. The described compoundsinhibit the overexpression of Her2, EGFR, Her3, and/or Her4 complexes.The discovered molecules, which are attainable for oral administration,can be used alone or in combination with cytotoxic drugs, hormonalagent, radiation, or other Her2 inhibitors to treat breast, lung,prostate, and other Her2-associated cancers. In addition, the discoveredcompounds exhibit great specificity against Her2/EGFR activity byselectively binding to only Her2 or Her2 associated dimers (for exampleHer2 homodimer, Her2-EGFR heterodimer, Her2-Her3 heterodimer andher2-her4 heterodimer). This might cause less severe side effects incomparison to the conventional tyrosine kinase inhibitors.

The small molecules describe by this invention can also be “tagged” withradio-isotopes for molecular imaging of tumor marker Her2 in vivo.Radio-labeled compounds are required for Positron Emission Tomography(PET) and Single Photon Emission Computed Tomography (SPECT). In vivomonitoring of the Her2 expressing tumor is useful in monitoring thedisease progression and assessing the treatment efficacy. Accordingly,the invention discloses a method of imaging an Her2 expressing tumorcomprising administering a marker to a patient having said Her2expressing tumor, said marker comprising a compound of Formula (I)containing an imaging enhancing agent.

Groups of compounds able to inhibit Her2/neu receptors are describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of screening, according to the procedure inExample 1, to show the effect of EGFR and Her2 inhibitors on T6-17 andSK-BR-3 cell proliferation. Data for compound #S22_(—)57 are highlighted(as “057”), showing reduced cellular growth in culture (p<0.05).

FIG. 2 shows representative illustrations of plates as developed fromExample 1. Again, plates for compound #S22_(—)57 are highlighted (as“057”), showing reduced cellular growth in culture relative to DMSOcontrol.

FIG. 3 shows the inhibition of cell proliferation by HER2/neuinhibitors, as described in Example 2.

FIG. 4. shows the inhibition of HER2 phosphorylation in HC1 cells, asdescribed in Example 3.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingFigures and Examples, which form a part of this disclosure. It is to beunderstood that this invention is not limited to the specific products,methods, conditions or parameters described and/or shown herein, andthat the terminology used herein is for the purpose of describingparticular embodiments by way of example only and is not intended to belimiting of any claimed invention. Similarly, any description as to apossible mechanism or mode of action or reason for improvement is meantto be illustrative only, and the invention herein is not to beconstrained by the correctness or incorrectness of any such suggestedmechanism or mode of action or reason for improvement. Throughout thistext, it is recognized that the descriptions refer both to the compoundsand to the resulting pharmaceutical compositions and methods ofmanufacture and use.

The present invention relates to use of small molecule compounds tomodulate or inhibit the activity of a receptor tyrosine kinase.

In various embodiments, the invention relates to a method of inhibitinga cell proliferative disorder characterized by over-activity and/orinappropriate activity of a protein tyrosine kinase portion of areceptor, especially an Her2 receptor, comprising administering to apatient in need of such treatment a pharmaceutically effective amount ofa compound of Formula (A), or a pharmaceutically acceptable salt orprodrug thereof:

where n is 1-5 and B is independently alkyl, hydroxy, halo,perfluoromethyl, carboxylic acid, ester, amide, substituted alkoxy,substituted thiol, substituted acyl, nitrile, or substituted aminogroup.

In certain embodiments, the invention teaches a method of inhibiting acell proliferative disorder characterized by over-activity and/orinappropriate activity of a receptor, including an erbB receptor,preferably an epidermal growth factor receptor (EGFR), or aplatelet-derived growth factor receptor, and/or the receptor is Her2,Her3, or Her4, preferably and Her2 receptor, comprising administering toa patient in need of such treatment a pharmaceutically effective amountof a compound of Formula (I), or a pharmaceutically acceptable salt orprodrug thereof:

or its tautomer

wherein X is independently at each occurrence a hydrogen, halo, hydroxy,carboxylic acid, ester, amide, substituted alkoxy, substituted thiol,substituted acyl, nitrile, or substituted amino group.

It should be appreciated that additional functionalization of thecompound can be achieved at both the alcohol-oxygen or amino-nitrogenpositions. The skilled artisan will appreciate that such substitutionsmay include alkylation or acylation and that the preferred site of thisfunctionalization depends on the nature of the functionalizing agent(e.g., selective protecting groups).

In other embodiments, the X moiety of the compound of Formula (I) ishalo, preferably independently chloro or iodo, or more preferably bothchloro:

which can also be represented as:

This latter compound is described within the Experimental section belowas SS22_(—)57, and is commercially available from Maybridge Chemicals,UK, Catalog #CD04155SC.

The skilled artisan will recognize that the substitutions of thearomatic halides of SS22_(—)57 with groups including hydrogen, halo,hydroxy, carboxylic acids, esters, amides, substituted alkoxy,substituted thiols, substituted acyl, nitriles, and substituted aminogroups. The latter allow for a wide range of X substituents from theSS22_(—)57 structure, all of which fall within the scope of thisinvention. For means to affect the specific transformations describedherein, see, e.g., J. March, Advanced Organic Chemistry, 2d ed., McGrawHill (1977), which is incorporated by reference in its entirety.

The sequences set forth herein are illustrative only, and are notintended to limit the scope of the invention. Those skilled in the artwill appreciate that modifications to the described synthetic schemescan be performed without detracting from the spirit of the invention.

The various substituents can be selected in view of factors such as, forexample, absorption, activity, affinity, distribution, excretion,metabolism, pharmacokinetic, solubility, toxicological and otherproperties conducive to their use as pharmaceuticals.

In further embodiments, the invention discloses a method of treating apatient having a disease characterized by over-activity and/orinappropriate activity of a receptor, including an erbB receptor,preferably an EGFR or a platelet-derived growth factor receptor, and/orthe receptor is Her2, Her3, or Her4, preferably and Her2 receptor,comprising the step of administering to said patient a pharmaceuticallyeffective amount of a compound having the Formula (I), of any of thepreviously cited chemical embodiments.

In still further embodiments, the invention teaches the use of acompound of any of the preceding chemical embodiments of Formula (I) forthe preparation of a medicament for the inhibition of a cellproliferative disorders characterized by over-activity and/orinappropriate activity of a receptor, including an erbB receptor,preferably an EGFR growth factor, or a platelet-derived growth factorreceptor, and/or the receptor is Her2, Her3, or Her4, preferably andHer2 receptor, and in still other embodiments, the invention discloses apharmaceutical composition comprising a compound of Formula (I) in anamount effective to inhibit a cell proliferative disorder characterizedby over-activity and/or inappropriate activity of a protein tyrosinekinase portion of a receptor, especially an Her2 receptor.

Each pharmaceutical compound embodiment may also be part of a commercialpackage, further comprising at least one anti-cancer agent and writteninstructions as to the use of the medicament. Such instructions mayinclude, for example, written matter stating that the pharmaceuticalcomposition can or should be used for the prophylaxis and/or treatmentof a disease caused by overexpression or activation of Her2 and/or EGFRand/or Her3 and/or Her4, specifics as to the disease being treated, anddose regimen recommendations.

In each of these embodiments, the ligand can include an epidermal growthfactor or a platelet-derived growth factor, and/or the tyrosine kinasereceptor is EGFR, Her2, Her3, or Her4 complexes such that the aim of themethod or composition is to inhibit the overexpression or activation ofHer2 and/or growth factors, thereby inhibiting associated diseases suchas cancer, angiogenesis associated with the growth of cancer or sarcoma,angiogenesis associated with cancer metastasis, angiogenesis associatedwith diabetic retinopathy, arteriosclerosis, restenosis, or psoriasis.”

Other specific examples of “a disease caused by overexpression oractivation of an erbB receptor, including Her2 and/or EGFR” includecancers such as brain tumor, pharyngeal cancer, laryngeal cancer, tonguecancer, esophageal cancer, gastric cancer, colorectal cancer, non-smallcell lung cancer, pancreatic cancer, bile duct cancer, gallbladdercancer, liver cancer, renal cancer, bladder cancer, prostate cancer,breast cancer, ovarian cancer, cervical cancer, endometrial cancer, skincancer, childhood solid cancer, bone tumor, hemangioma and the like,angiogenesis associated with diabetic retinopathy, arteriosclerosis,psoriasis and the like. Preferred are brain tumor, pharyngeal cancer,laryngeal cancer, tongue cancer, esophageal cancer, gastric cancer,colorectal cancer, non-small cell lung cancer, pancreatic cancer, bileduct cancer, gallbladder cancer, liver cancer, renal cancer, bladdercancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer,endometrial cancer, skin cancer and the like and more preferred arebrain tumor, gastric cancer, colorectal cancer, non-small cell lungcancer, pancreatic cancer, renal cancer, prostate cancer, breast cancer,ovarian cancer and the like.

In separate embodiments, the patient being treated or to whom thepharmaceutical composition is to be applied is a human.

This invention can be applied to treat cell proliferative disorders,such as cancers characterized by the over-activity or inappropriateactivity of erbB receptors, when used alone or in combination withchemotherapy (e.g., cytotoxic chemotherapy or radiation or both) to haltresistance to currently available drugs. The invention may also beapplied in combination with administering a pharmaceutically effectiveamount of an anti-cancer agent or performing a non-drug therapy or bothon the patient, where the embodied compounds are administered at thesame or different times as other anti-cancer agents, and/or where thenon-drug therapy is surgery, hypertensive chemotherapy, genetherapy,thermotherapy, cryotherapy, photodynamic therapy, laser cauterizationand/or radiotherapy.

The invention provides a novel class of Her2 and EGFR inhibitors thatmight halt the resistance to current tyrosine kinase inhibitors henceincrease treatment efficacy, and as such provide an alternative to theuse of current tyrosine kinase inhibitors. The discovered molecules,which are attainable for oral administration, can be used alone or incombination with cytotoxic drugs, hormonal agent, radiation, or otherHer2 inhibitors to treat breast, lung, prostate, and otherHer2-associated cancers. In addition, the discovered compounds exhibitgreat specificity against Her2/EGFR activity by selectively binding toonly Her2 or Her2 associated dimers (for example Her2 homodimer,Her2-EGFR heterodimer, Her2-Her3 heterodimer and her2-her4 heterodimer).This might cause less severe side effects in comparison to theconventional tyrosine kinase inhibitors.

The small molecules describe by this invention can also be “tagged” withradio-isotopes for molecular imaging of tumor marker Her2 in vivo.Radio-labeled compounds are required for Positron Emission Tomography(PET) and Single Photon Emission Computed Tomography (SPECT). In vivomonitoring of the Her-2 expressing tumor is useful in monitoring thedisease progression and assessing the treatment efficacy. Accordingly,the invention discloses a method of imaging an Her-2 expressing tumorcomprising administering a marker to a patient having said Her-2expressing tumor, said marker comprising a compound of Formula (I)containing an imaging enhancing agent.

In the present disclosure the singular forms “a,” “an,” and “the”include the plural reference, and reference to a particular numericalvalue includes at least that particular value, unless the contextclearly indicates otherwise. Thus, for example, a reference to “acompound” is a reference to at least one of such compounds andequivalents thereof known to those skilled in the art, and so forth.

When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. In general, use of the term “about” indicates approximationsthat can vary depending on the desired properties sought to be obtainedby the disclosed subject matter and is to be interpreted in the specificcontext in which it is used, based on its function, and the personskilled in the art will be able to interpret it as such. Where present,all ranges are inclusive and combinable.

It is to be appreciated that certain features of the invention whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, reference to values statedin ranges include each and every value within that range.

Generally terms are to be given their plain and ordinary meaning such asunderstood by those skilled in the art, in the context in which theyarise. To avoid any ambiguity, however, several terms are describedherein.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences that are provided throughout this document. The nomenclatureused herein and the laboratory procedures in analytical chemistry, andorganic synthetic chemistry described below are those well known andcommonly employed in the art. Standard techniques, or modificationsthereof, are used for chemical syntheses and chemical analyses.

Whenever a group of this invention is described as being “optionallysubstituted” that group may be unsubstituted or substituted with one ormore of the substituents described for that group. Likewise, when agroup is described as being “unsubstituted or substituted,” ifsubstituted, the substituent may be selected from the same group ofsubstituents. Unless otherwise indicated, when a substituent is deemedto be “optionally substituted,” or “substituted” it is meant that thesubstituent is a group that may be substituted with one or more group(s)individually and independently selected.

Each of the following terms (e.g., “alkyl,” “heteroalkyl,” “acyl,”“alkoxy,” “aryl,” and “heteroaryl”) include both substituted andunsubstituted forms of the indicated group, unless indicated otherwise.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon group (cycloalkyl), or combination thereof, which may befully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, and can have a number of carbon atoms optionallydesignated (e.g., C₁₋₃ means one to three carbons). Examples ofsaturated hydrocarbon groups include, but are not limited to, groupssuch as methyl, ethyl, n-propyl, isopropyl.

An alkyl group of this invention may be substituted or unsubstituted.When substituted, the substituent group(s) may be one or more group(s)independently selected from cycloalkyl, aryl, heteroaryl,heteroalicyclyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, cyano, halo, oxo, carbonyl, thiocarbonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido,S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato,thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, aminoor substituted amino, protected hydroxyl, protected amino, protectedcarboxy and protected amido groups.

As used herein, “acyl” refers to an “RC(═O)—.” An acyl group may containan alkyl or aryl moiety, in which case it may be referred to as acarboxyalkyl or carboxyaryl group, respectively. Examples of acyl groupsinclude, without limitation, formyl, acetyl, propionyl, butyryl,pentanoyl, pivaloyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl,undecanoyl, dodecanoyl and benzoyl. Presently preferred acyl groups areacetyl and benzoyl.

An acyl group of this invention may be unsubstituted or substituted.When substituted, the substituent(s) may be selected from the samegroups disclosed above with regard to alkyl group substitution.

The term “alkoxy” is used in its conventional sense, and refers to thosealkyl groups attached to the remainder of the molecule via an oxygenatom. Alkoxy groups include, but are not limited to methoxy, ethoxy,propoxy, trifluoromethoxy and difluoromethoxy.

An “amide” is a chemical moiety with formula —(R)_(n)—C(O)NHR′ or—(R)_(n)—NHC(O)R′, where n is the number of alkylene carbons from 0-1, Rand R′ are independently selected from the group consisting of alkyl,cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) andheteroalicyclic (bonded through a ring carbon). An amide may be an aminoacid or a peptide molecule attached to a molecule of the presentinvention, thereby forming a prodrug.

Any amine, hydroxy, or carboxyl side chain on the compounds of thepresent invention can be esterified or amidified. The procedures andspecific groups to be used to achieve this end is known to those ofskill in the art and can readily be found in reference sources such asGreene and Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed.,John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein inits entirety.

The term “ester” refers to a chemical moiety with formula—(R)_(n)—COOR′, where R and R′ are independently selected from the groupconsisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ringcarbon) and heteroalicyclic (bonded through a ring carbon), and where nis 0 or 1.

As used herein, an “ether” refers to an “—C—O—C—” group wherein eitheror both carbons may independently be part of an alkyl, alkenyl, alkynyl,aryl, heteroaryl or heteroalicyclyl group. A “halogenated ether” refersto an ether in which the groups to either side of the oxygen are bothalkyl substituted with halogen.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Preferred halogens are chloro, iodo, andfluoro.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon group, or combinations thereof, consisting of anumber of carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, Si and S, and wherein the nitrogen, carbon andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), carbon-11 (¹¹C), carbon-14 (¹⁴C), nitrogen-13(¹³N), sulfur-35 (³⁵S), iodine-131 (¹³¹I), or fluorine-18 (¹⁸F). Whenindicated as such, radiolabled molecules contain isotopes enriched abovetheir natural abundances. Such radioisotopes can be prepared either bysynthetic means using commercially available precursors and reactantsgenerally recognized as providing by substitution the particular elementof interest (e.g., halide substitution using radioactive halides) or bycyclotron irradiation of the pre-formed molecules. All isotopicvariations of the compounds of the present invention, whetherradioactive or not, are intended to be encompassed within the scope ofthe present invention.

As used herein, “phenyl” refers to a 6-member aryl group. A phenyl groupmay be unsubstituted or substituted. When substituted the substituent(s)is/are one or more, preferably one or two, group(s) independentlyselected from the group consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, alkyl, alkoxy, acyl, acyloxy, carboxy, protectedcarboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl,protected hydroxymethyl, amino or substituted amino, carboxamide,protected carboxamide, N-alkylcarboxamide, protected N-alkylcarboxamide,N,N-dialkylcarboxamide, trifluoromethyl, N-alkylsulfonylamino,N-(phenylsulfonyl)amino and phenyl (resulting in the formation of abiphenyl group).

As used herein, “amino protecting group” refers to a group commonlyemployed to keep (i.e., to “block” or “protect”) an amino group fromreacting with a reagent while it reacts with an intended targetfunctional group of a molecule.

Examples of amino protecting groups include, without limitation, formyl(“For”), trityl, phthalimido, trichloroacetyl, chloroacetyl,bromoacetyl, iodoacetyl groups, t-butoxycarbonyl (“Boc”),2-(4-biphenylyl)propyl-2-oxycarbonyl (“Bpoc”),2-phenylpropyl-2-oxycarbonyl (“Poc”), 2-(4-xenyl)isopropoxycarbonyl,1,1-diphenylethyl-1-oxycarbonyl, 1,1-diphenylpropyl-1-oxycarbonyl,2-(3,5-dimethoxyphenyl)propyl-2-oxycarbonyl (“Ddz”),2-(p-toluoyl)propyl-2-oxycarbonyl, cyclopentanyloxycarbonyl,1-methylcyclopentanyloxycarbonyl, cyclohexanyloxy-carbonyl,1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl,2-(4-toluoylsulfonyl)-ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl,2-(triphenylphosphino)-ethoxycarbonyl, 9-fluorenylmethoxycarbonyl(“Fmoc”), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl,1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyl-oxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropyl-methoxycarbonyl, isobornyloxycarbonyl,1-piperidyloxycarbonyl, benzyloxycarbonyl (“Cbz”),4-phenylbenzyloxycarbonyl, 2-methylbenzyloxy-carbonyl,-2,4,5-tetramethylbenzyloxycarbonyl (“Tmz”),4-methoxybenzyloxy-carbonyl, 4-fluorobenzyloxycarbonyl,4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyl-oxycarbonyl,4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,4-nitrobenzyloxy-carbonyl, 4-cyanobenzyloxycarbonyl,4-(decyloxy)benzyloxycarbonyl, benzoylmethylsulfonyl, dithiasuccinoyl(“Dts”), 2-(nitro)phenylsulfenyl (“Nps”), and diphenyl-phosphine oxide.The species of amino-protecting group employed is not critical so longas the derivatized amino group is stable to the conditions of thesubsequent reaction(s) and can be removed at the appropriate pointwithout disrupting the remainder of the molecule. Presently preferredamino-protecting groups are Boc, Cbz and Fmoc. Descriptions of these andother amino-protecting groups may be found in T. W. Greene and P. G. M.Wuts, “Protective Groups in Organic Synthesis,” 2nd ed., John Wiley andSons, New York, N.Y., 1991, Chapter 7, M. Bodanzsky, “Principles ofPeptide Synthesis,” 1st and 2nd revised ed., Springer-Verlag, New York,N.Y., 1984 and 1993, and Stewart and Young, “Solid Phase PeptideSynthesis,” 2nd ed., Pierce Chemical Co., Rockford, Ill., 1984.

As used herein, the term “carboxy protecting group” refers to a labileester commonly used to block or protect a carboxylic acid whilereactions are carried out on other functional groups on the compound.Examples of carboxy protecting groups include, without limitation,t-butyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl,2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl,pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl,4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, 2-phenylpropyl,trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl,-(trimethylsilyl)ethyl, -(di(n-butyl)methylsilyl)ethyl,p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl, and1-(trimethylsilylmethyl)-propenyl. The ester employed is not critical solong as it is stable to the conditions of subsequent reaction(s) and canbe removed at the appropriate point without disrupting the remainder ofthe molecule. Further examples of carboxy-protecting groups are found inE. Haslam, “Protective Groups in Organic Chemistry,” J. G. W. McOmie,Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W. Greene andP. G. M. Wuts, “Protective Groups in Organic Synthesis,” 2nd ed., JohnWiley and Sons, New York, N.Y., 1991, Chapter 5.

As used herein, a “hydroxyl protecting group” refers to a readilycleavable group that replaces the hydrogen of the hydroxyl group, suchas, without limitation, tetrahydropyranyl, 2-methoxypropyl,1-ethoxyethyl, methoxymethyl, 2-methoxyethoxymethyl, methylthiomethyl,t-butyl, t-amyl, trityl, 4-methoxytrityl, 4,4′-dimethoxytrityl,4,4′,4″-trimethoxytrityl, benzyl, allyl, trimethylsilyl,(t-butyl)dimethylsilyl, and 2,2,2-trichloroethoxycarbonyl. The speciesof hydroxyl protecting groups is not critical so long as the derivatizedhydroxyl group is stable to the conditions of subsequent reaction(s) andcan be removed at the appropriate point without disrupting the remainderof the molecule. Further examples of hydroxy-protecting groups aredescribed by C. B. Reese and E. Haslam, “Protective Groups in OrganicChemistry,” J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973,Chapters 3 and 4, respectively, and T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis,” 2nd ed., John Wiley and Sons,New York, N.Y., 1991, Chapters 2 and 3.

By “perhaloalkyl” it is meant an alkyl moiety where all of the hydrogenatoms normally present on the alkyl are replaced by a halogen. Thus, forexample, a perchloroalkyl is an alkyl moiety where all of the carbonatoms not connected to the rest of the molecule are connected tochlorine atoms.

Throughout the present disclosure, when a particular compound comprisesa chiral center, the scope of the present disclosure also includescompositions comprising the racemic mixture of the two enantiomers, aswell as compositions comprising each enantiomer individuallysubstantially free of the other enantiomer. Thus, for example,contemplated herein is a composition comprising the S enantiomersubstantially free of the R enantiomer, or a composition comprising theR enantiomer substantially free of the S enantiomer. By “substantiallyfree” it is meant that the composition comprises less than 10%, or lessthan 8%, or less than 5%, or less than 3%, or less than 1% of the minorenantiomer. If the particular compound comprises more than one chiralcenter, the scope of the present disclosure also includes compositionscomprising a mixture of the various diastereomers, as well ascompositions comprising each diastereomer substantially free of theother diastereomers. The recitation of a compound, without reference toany of its particular diastereomers, includes compositions comprisingall four diastereomers, compositions comprising the racemic mixture ofR,R and S,S isomers, compositions comprising the racemic mixture of R,Sand S,R isomers, compositions comprising the R,R enantiomersubstantially free of the other diastereomers, compositions comprisingthe S,S enantiomer substantially free of the other diastereomers,compositions comprising the R,S enantiomer substantially free of theother diastereomers, and compositions comprising the S,R enantiomersubstantially free of the other diastereomers.

Inhibiting Cell Proliferative Disorders

The term “therapeutic agent” is intended to mean a compound that, whenpresent in a therapeutically effective amount, produces a desiredtherapeutic effect on a patient. For example, an “effective amount” of acompound for inhibiting Her2/neu action is an amount of a compound orcomposition that is sufficient to inhibit, reduce, or otherwise mitigatean undesirable effect of Her2/neu action. Such inhibition may occur forexample, and without limitation, via a direct interaction, and/orthrough a competitive interaction, or via an allosteric interaction witha corresponding receptor.

Medical Imaging

These small molecules can also be conjugated, or “tagged” withfluorescent probes or radio-isotopes for molecular imaging of tumormarker Her2 in vivo. Long wavelength fluorescent probes are suitable foroptical imaging, while radio labeled compounds are required for PositronEmission Tomography (PET) and Single Photon Emission Computed Tomography(SPECT).

Positron Emission Tomography (PET) is a precise and sophisticated,non-invasive technique using isotopes produced in a cyclotron. Apositron-emitting radionuclide is introduced, usually by injection, andaccumulates in the target tissue. As it decays it emits a positron,which promptly combines with a nearby electron resulting in thesimultaneous emission of two identifiable gamma rays in oppositedirections. These are detected by a PET camera and give very preciseindication of their origin.

Salts and Derivatives

The term “pharmaceutically acceptable salt” means those salts ofcompounds of the invention that are safe and effective for use in asubject and that possess the desired biological activity.Pharmaceutically acceptable salts include salts of acidic or basicgroups present in compounds of the invention. Various pharmaceuticallyacceptable salts, ether derivatives, ester derivatives, acidderivatives, and aqueous solubility altering derivatives of the activecompound also are encompassed by the present invention. The presentinvention further includes all individual enantiomers, diastereomers,racemates, and other isomers of the compound. The invention alsoincludes all polymorphs and solvates, such as hydrates and those formedwith organic solvents, of this compound. Such isomers, polymorphs, andsolvates may be prepared by methods known in the art, such as byregiospecific and/or enantioselective synthesis and resolution, based onthe disclosure provided herein.

As the compounds of the invention may contain charged side chains ortermini, they may be included in any of the above-described formulationsas the free acids or bases or as pharmaceutically acceptable salts.Pharmaceutically acceptable salts are those salts which substantiallyretain the physiological activity of the free bases and which areprepared by reaction with inorganic acids. Pharmaceutical salts tend tobe more soluble in aqueous and other protic solvents than are thecorresponding free base forms.

Suitable salts of the compound include, but are not limited to, acidaddition salts, such as those made with hydrochloric, hydrobromic,hydroiodic, hydrofluoric, perchloric, sulfuric, nitric, phosphoric,acetic, propionic, glycolic, lactic pyruvic, malonic, succinic, maleic,fumaric, malic, tartaric, citric, benzoic, carbonic cinnamic, mandelic,methanesulfonic, ethanesulfonic, hydroxyethanesulfonic,benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic,p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; saltsmade with saccharin; alkali metal salts, such as lithium, sodium, andpotassium salts; alkaline earth metal salts, such as calcium andmagnesium salts; salts formed from Lewis acids, such as borontrifluoride; and salts formed with organic or inorganic ligands, such asquaternary ammonium salts (for example, tris(hydroxymethyl)aminomethanesalts).

Additional suitable salts include, but are not limited to, acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate,malate, maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammoniumsalt, oleate, pamoate (embonate), palmitate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate,subacetate, succinate, tannate, tartrate, teoclate, tosylate,triethiodide and valerate salts of the compound of the presentinvention. For a review on pharmaceutically acceptable salts see Bergeet al., 66 J. Pharm. Sci 1-19 (1977), incorporated herein by reference.

Prodrugs and active metabolites of compounds disclosed herein are alsowithin the scope of the invention.

A prodrug is a pharmacologically inactive compound that is convertedinto a pharmacologically active agent by a metabolic transformation orany other chemical or biological process (e.g., hydrolysis). Forexample, in vivo, a prodrug can be acted on by naturally occurringenzyme(s) resulting in liberation of the pharmacologically active agent.Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. An example, without limitation, of a prodrug wouldbe a compound of the present invention which is administered as an ester(the “prodrug”) to facilitate transmittal across a cell membrane wherewater solubility is detrimental to mobility but which then ismetabolically hydrolyzed to the carboxylic acid, the active entity, onceinside the cell where water-solubility is beneficial. A further exampleof a prodrug might be a short peptide (polyaminoacid) bonded to an acidgroup where the peptide is metabolized to reveal the active moiety.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs,”ed. H. Bundgaard, Elsevier, 1985.

An active metabolite is a compound that results from metabolism ofanother compound after administration of the latter to a subject.Metabolites can be identified by techniques well-known in the art.

Formulation and Administration

The compounds of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat, inhibit, orprevent Her2-mediated cell proliferative disorders, the compounds of theinvention, or pharmaceutical compositions thereof, are administered orapplied in a therapeutically effective amount. By “therapeuticallyeffective amount” is meant an amount effective ameliorate or prevent thesymptoms, prolong the survival of, or otherwise mitigate the undesirableeffects of the disease for which the patient is being treated.Determination of a therapeutically effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure provided herein. The term “prevent” as used hereinto describe the action of inhibiting cell proliferation or the growth oftumors, or ameliorating the symptoms, prolonging the survival of, orotherwise mitigating the undesirable effects of the disease for whichthe patient is being treated.

For detection of expression or activity of Her2 and/or EGFR, a tissue(cancer tissue, blood vessel wall tissue, skin, oral mucosa etc.) or abody fluid (blood, lymph) and the like, which is obtained from patients,is applied to a test to detect expression or activity of Her2 and/orEGFR. Such tests are known to those skilled in the art. With regard tothe treatment of some conditions, for example, atherosclerosis, certainpeople may be identified as being at high risk, for example, due togenetic, environmental or historical factors. Compounds within the scopeof the present invention can be used in preventing or delaying theoccurrence or reoccurrence of such conditions or otherwise treating thecondition.

As mentioned above, the inhibitor of the present invention is aneffective treatment method of cancer patients and also expected to be anagent for the prophylaxis and/or treatment of preventing transition fromhormone sensitive cancer to resistant cancer in prostate cancer andbreast cancer. Moreover, it is expected to an agent for the prophylaxisand/or treatment of angiogenesis associated with the growth of solidcancer and sarcoma, angiogenesis associated with cancer metastasis,angiogenesis associated with diabetic retinopathy, arteriosclerosis,psoriasis and the like.

The “overexpression or activation of Her2 and/or EGFR” is an expressionor activity not less than the expression amount or activity necessaryfor homeostasis of living organisms, and the expression or activity notless than the expression amount or activity necessary for normal tissueof the same origin.

The “patients showing overexpression or activation of Her2 and/or EGFR”means the patients wherein at least one of Her2 and EGFR is excessivelyexpressed or activated, and preferably the patients wherein both areexcessively expressed or activated. The Her2 and/or EGFR inhibitor ofthe present invention is characterized by administration for thetreatment of patients, wherein Her2 and/or EGFR are/is excessivelyexpressed or activated as mentioned above.

The “Her2 and/or EGFR inhibitor” of the present invention is preferablya Her2 and EGFR inhibitor to be administered to patients wherein Her2and EGFR are excessively expressed or activated, and may be a mixture ofa Her2 inhibitor and an EGFR inhibitor. It is possible to use a Her2inhibitor and an EGFR inhibitor simultaneously, separately or at timeintervals. In other words, it is possible to administer a Her2 inhibitorand an EGFR inhibitor simultaneously, separately or, for example, in astaggered manner in a single day or at given time intervals for severaldays to several weeks or several months, by various different routes.

The amount of compound administered will, of course, be dependent on thesubject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician. However, to the extent that these compoundsprovide improved activity relative to other known small molecules in invivo, in vitro, and animal studies, in the broadest sense, recommendeddosages are those similar to those currently prescribed for other smallmolecules for this same purpose.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable. The therapy may be provided alone orin combination with other drugs.

Preferably, a therapeutically effective dose of the compounds describedherein will provide therapeutic benefit without causing substantialtoxicity.

Toxicity of the compounds described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use human. The dosageof the compounds described lies preferably within a range of circulatingconcentrations that include the effective dose with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p. 1). Preferred dosages range from 1 nM to 500 mM.

Suitable dosage forms include but are not limited to oral, rectal,sub-lingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular,intravenous, transdermal, spinal, intrathecal, intra-articular,intra-arterial, sub-arachinoid, bronchial, lymphatic, and intra-uterilleadministration, and other dosage forms for systemic delivery of activeingredients. In a preferred embodiment, the dosage form is suitable fororal administration.

To prepare such pharmaceutical dosage forms, one or more of theaforementioned compounds or a pharmaceutically acceptable salt thereof,are intimately admixed with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of preparation desired foradministration.

The compounds of the invention, may be administered to a subject per seor in the form of a pharmaceutical composition. Pharmaceuticalcompositions comprising the compounds of the invention may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the active peptides or peptide analogues into preparationswhich can be used pharmaceutically. Proper formulation is dependent uponthe route of administration chosen.

For parenteral formulations, the carrier will usually comprise sterilewater, though other ingredients, for example, ingredients that aidsolubility or for preservation, may be included. Injectable solutionsmay also be prepared in which case appropriate stabilizing agents may beemployed.

Parenteral administration may comprise any suitable form of systemicdelivery or delivery directly to the CNS. Administration may for examplebe intravenous, intra-arterial, intrathecal, intramuscular,subcutaneous, intramuscular, intra-abdominal (e.g., intraperitoneal),etc., and may be effected by infusion pumps (external or implantable) orany other suitable means appropriate to the desired administrationmodality.

In preparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed. Thus, the compounds can be readilyformulated by combining the compounds, salts, or analogues withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.

In some applications, it may be advantageous to utilize the active agentin a “vectorized” form, such as by encapsulation of the active agent ina liposome, micelle, or other encapsulant medium, or by fixation of theactive agent, e.g., by covalent bonding, chelation, assembly, orassociative coordination, on a suitable biomolecule, such as thoseselected from proteins, lipoproteins, glycoproteins, andpolysaccharides.

For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, such aslactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For solid oral preparations such as, for example, powders, capsules,caplets, and tablets, suitable carriers and additives include starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like. Due to their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform. If desired, tablets may be sugar coated or enteric coated bystandard techniques.

Treatment methods of the present invention using formulations suitablefor oral administration may be presented as discrete units such ascapsules, cachets, tablets, or lozenges, each containing a predeterminedamount of the active ingredient as a powder or granules. Optionally, asuspension in an aqueous liquor or a non-aqueous liquid may be employed,such as a syrup, an elixir, an emulsion, or a draught.

A tablet may be made by compression or molding, or wet granulation,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine, with the activecompound being in a free-flowing form such as a powder or granules whichoptionally is mixed with a binder, disintegrant, lubricant, inertdiluent, surface active agent, or discharging agent. Molded tabletscomprised of a mixture of the powdered active compound with a suitablecarrier may be made by molding in a suitable machine.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredient(s). Such accessory ingredient(s) mayinclude flavorings, suitable preservative, agents to retardcrystallization of the sugar, and agents to increase the solubility ofany other ingredient, such as a polyhydroxy alcohol, for exampleglycerol or sorbitol.

For buccal administration, the compounds may take the form of tablets,lozenges, etc. formulated in conventional manner.

For topical administration the compounds of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

Formulations suitable for parenteral administration usually comprise asterile aqueous preparation of the active compound, which preferably isisotonic with the blood of the recipient (e.g., physiological salinesolution). Such formulations may include suspending agents andthickening agents and liposomes or other microparticulate systems whichare designed to target the compound to blood components or one or moreorgans. The formulations may be presented in unit-dose or multi-doseform.

Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

Nasal and other mucosal spray formulations (e.g., inhalable forms) cancomprise purified aqueous solutions of the active compounds withpreservative agents and isotonic agents. Such formulations arepreferably adjusted to a pH and isotonic state compatible with the nasalor other mucous membranes. Alternatively, they can be in the form offinely divided solid powders suspended in a gas carrier. Suchformulations may be delivered by any suitable means or method, e.g., bynebulizer, atomizer, metered dose inhaler, or the like.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from pressurized packs or a nebulizer, with the use of apropellant acceptable as suitable by the pharmaceutical industry.Suitable propellants include, but are not limited todichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane, P-227ea, carbondioxide or other suitable gas. In the case of a pressurized aerosol thedosage unit may be determined by providing a valve to deliver a meteredamount. Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Alternatively, the compounds may be in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides, hydrogenatedfats, or hydrogenated fatty carboxylic acids.

Transdermal formulations may be prepared by incorporating the activeagent in a thixotropic or gelatinous carrier such as a cellulosicmedium, e.g., methyl cellulose or hydroxyethyl cellulose, with theresulting formulation then being packed in a transdermal device adaptedto be secured in dermal contact with the skin of a wearer.

In addition to the aforementioned ingredients, formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, binders,disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants), and the like. Suchpharmaceutical compositions can be prepared by methods and containcarriers which are well-known in the art. A generally recognizedcompendium of such methods and ingredients is Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippingcottWilliams and Wilkins: Philadelphia, Pa., 2000.

The formulation of the present invention can have immediate release,sustained release, delayed-onset release or any other release profileknown to one skilled in the art.

The subject receiving the pharmaceutical composition is preferably ananimal, including, but not limited, to an animal such a cow, horse,sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, andguinea pig, and is more preferably a mammal, and most preferably ahuman.

The amount of the active agent to be administered can typically rangefrom between about 0.01 to about 25 mg/kg/day, preferably from betweenabout 0.1 to about 10 mg/kg/day and most preferably from between about0.2 to about 5 mg/kg/day. It will be understood that the pharmaceuticalformulations of the present invention need not necessarily contain theentire amount of the agent that is effective in treating the disorder,as such effective amounts can be reached by administration of aplurality of doses of such pharmaceutical formulations.

In a preferred embodiment of the present invention, the compounds areformulated in capsules or tablets, preferably containing 25 to 200 mg ofthe compounds of the invention, and are preferably administered to apatient at a total daily dose of about 0.5 mg to about 2 g, preferablyabout 7.5 mg to about 750 mg, more preferably about 15 mg to 750 mg, andmost preferably from about 50 to about 200 mg.

A pharmaceutical composition for parenteral administration contains fromabout 0.01% to about 100% by weight of the active agents of the presentinvention, based upon 100% weight of total pharmaceutical composition.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver peptides and peptide analogues of theinvention. Certain organic solvents such as dimethylsulfoxide also maybe employed, although usually at the cost of greater toxicity.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5to 1 mg/kg/day. Therapeutically effective serum levels may be achievedby administering multiple doses each day. Some preferred dosages rangefrom 1 nM to 500 mM. Some preferred dosages range from 1 mM to 500 mM.Some preferred dosages range from 1 mg to 500 mg. Some preferred dosagesrange from 1000 mg to 3000 mg. Some preferred dosages range from 1500 mgto 2500 mg.

In cases of local administration or selective uptake, the effectivelocal concentration of the compounds may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

EXPERIMENTAL Example 1 Screening Studies

Targeted molecules were screened in vitro for anti-tumor activity usinga modified anchorage-dependent growth of transformed cell assay, asdescribed in Fukazawa et al. (1995), Anal. Biochem., 10:83-90.Anchorage-independent growth capability is perhaps the best indicator ofthe malignant phenotype and is determined by efficiency of cells to formcolonies in soft agar (Drebin, et al., Cell 41, 697-706 (1985)). Allexperiments were conducted in 6 cm dishes in triplicate. Agar bottomlayers consisted of 3 ml 0.25% agarose/DMEM (Dulbecco's Modified EagleMedium) supplemented with 10% FBS (Fetal Bovine Serum), 2 mML-glutamine, and 100 u/ml Penicillin/1 μg/ml Streptomycin. About1000-2000 cells T6-17/SK-BR-3 are layered on the top in 1 ml 0.18%agarose/DMEM, 10% FBS, 2 mM L-glutamine, and 100 μ/ml Penicillin/1 μg/mlStreptomycin. Cultures were treated every 3 days with 0.5 ml ofsupplemented DMEM containing measured amounts of the Her2 inhibitors, orDMSO (dimethyl sulfoxide) alone. On the day before the colonies werecounted, the samples were incubated with 1 ml of Hank's Balanced SaltSolution containing 1 mg/ml p-iodonitrotetrazolium violet (INT, Sigma)to stain colonies. The next day, colonies were counted using a 7×achromatic magnifying eyepiece (Bausch and Lomb). Triplicate cultureswere counted on day 21 and will be verified by at least two differentpersons. Bars represent the mean values±one standard deviation.

The results of this testing is shown in FIG. 1. FIG. 2 showsrepresentative plates for the individual compounds.

Example 2 Inhibition of Proliferation of Several Cell Lines

The activity of S22-57 in cell proliferation assays using different celllines was examined. T6-17 is a murine cell line engineered to expresshuman HER2/neu. HC1 is a human non-small lung cancer cell line withupregulated HER2/neu and resistant to anti-EGFR antibody cetuximab.HBL100 is an immortalized human breast cancer cell line. XHT-1-1A aremouse NIH3T3 cells transformed by Harvey-RAS (XHT-1-1a) previouslydeveloped and used (Drebin, et al., Cell 41, 697-706 (1985)). BothHBL100 and XHT-1-1A are used as controls. To measure cell proliferation,we used the modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (Masuda, et al.,Oncogene 25, 7740-7746 (2006)). Cells (2000-5000 cells/well) were seededin 96-well plates in the presence of inhibitors with differentconcentration and incubated at 37° C. in a humidified 5% CO2 atmosphere.After 72 h, cells were incubated in fresh medium for another 4 hoursbefore a total of 25 μl of MTT solution (5 mg/ml in PBS) was added toeach well, and after 2 h of incubation at 37° C., 100 μl of theextraction buffer (20% w/v SDS, 50% N,N-dimethyl formamide, pH 4.7) wasadded. After an overnight incubation at 37° C., the optical density at570 nm was measured using a Tecan ELISA reader. FIG. 3 shows the resultsof experiments in which cells were treated with inhibitors at indicateddoses for three days. Proliferation of cells was measured by the MTTassay. As shown in FIG. 3, S22-05 at 10 μg/ml selectively inhibited theproliferation of T6-17 and HC1 cells. S22-57 also inhibited T6-17 andHC1 dose-dependently.

Example 3 Inhibition of HER2 Phosphorylation in HC1 Cells

The effect of S22-57 was also examined in the cetuximab-resistantnon-small cell lung cancer cell line HC1. Confluent HC1 cells were firstserum starved for 24 hours before exposed to inhibitors as indicated for1 hour. Cells were then stimulated by EGF (50 ng/ml) for 10 minutes.Cell lysates were collected for western blot using antibodies toHER2Y877 or p-AKT, following protocols suggested by manufacturers. Bothantibodies were purchased from Cell Signaling. As shown in FIG. 4,S22-57 inhibited EGF-stimulated phosphorylation of HER2 at Y877.

What is claimed:
 1. A method of inhibiting a cell proliferative disordercharacterized by over-activity and/or inappropriate activity of Her2,the method comprising administering to a patient in need of suchtreatment a pharmaceutically effective amount of a compound of Formula(I), or a pharmaceutically acceptable salt or prodrug thereof:

wherein X is independently at each occurrence alkyl, halo,perfluoromethyl, hydroxy, carboxylic acid, ester, amide, substitutedalkoxy, substituted thiol, substituted acyl, nitrile, or substitutedamino group.
 2. The method of claim 1 wherein X is halo.
 3. The methodof claim 1 wherein the compound of Formula (I) is


4. The method of claim 1 wherein the cell proliferative disorder iscancer.
 5. The method of claim 1 wherein the cell proliferative disorderaffects a breast, prostate, lung, pancreas, ovary, or stomach.
 6. Themethod of claim 1 wherein the patient is a human.
 7. The method of claim1 further comprising administering a pharmaceutically effective amountof an anti-cancer agent or performing a non-drug therapy or both to thepatient.
 8. The method of claim 7 wherein the compound of Formula (I)and the anti-cancer agent are administered at the same time.
 9. Themethod of claim 7 wherein the non-drug therapy is surgery, hypertensivechemotherapy, genetherapy, thermotherapy, cryotherapy, photodynamictherapy, laser cauterization and/or radiotherapy.
 10. A pharmaceuticalcomposition comprising a compound of Formula (I), or a pharmaceuticallyacceptable salt or prodrug thereof, in an amount effective to inhibit acell proliferative disorder characterized by over-activity and/orinappropriate activity of Her2, and a pharmaceutically acceptableexcipient:

wherein X is independently at each occurrence an alkyl, halo,perfluoromethyl, hydroxy, carboxylic acid, ester, amide, substitutedalkoxy, substituted thiol, substituted acyl, nitrile, or substitutedamino group.
 11. The composition of claim 10 wherein X is halo.
 12. Thecomposition of claim 10 wherein the compound of Formula (1) is


13. The composition of claim 10 wherein the cell proliferative disorderis cancer.
 14. The composition of claim 10 wherein the cellproliferative disorder affects a breast, prostate, lung, pancreas,ovary, or stomach.
 15. The composition of claim 10 wherein the cellproliferative disorder is psoriasis, atherosclerosis or restenosis. 16.The composition of claim 10 wherein the patient is a human.
 17. Acommercial package comprising the pharmaceutical composition of claim10, at least one anti-cancer agent, and a written matter associatedtherewith, the written matter stating that the pharmaceuticalcomposition can or should be used for treatment of a disease caused byoverexpression or activation of Her2.